Classical drug discovery to a large extent relies on animal models of disease, mostly in rodents. The reasons for this are manifold, but in part they center on practical aspects such as availability and cost, compounded by ethical considerations. Mice and rats are most frequently used, and many diseases have been modeled in these species. One question that has of late received more attention is that of species variability. Even if the disease is modeled well in the animal, variability in the drug target between humans and rodents may still lead to failed human trials, a venture that is both costly and time-consuming. One example of this is 12/15-lipoxygenase (12/15-LOX), an enzyme, which contributes to ischemic brain injury in both humans and mice.
The lipoxygenases form a large family of enzymes capable of oxidizing arachidonic acid (AA) and related polyunsaturated fatty acids (Brash A R, J. Biol. Chem. 1999, 274, 23679-23682). In humans, in addition to 12/15-LOX, other members include 5-LOX, P-12-LOX, 12(R)-LOX, epidermal LOX-3, and 15-LOX-2. Their nomenclature is based in part upon the tissue where they were first detected, and in part upon the carbon atom in AA that is oxidized. Correspondingly, 12/15-LOX can oxidize both C12 and C15, forming 12- or 15-hydroperoxyarachidonic acid (12- or 15-HPETE), respectively. Human lipoxygenases and their metabolites have been implicated in numerous diseases. 5-LOX has been implicated in cancer, asthma, COPD, allergic rhinitis, osteoarthritis, and atherosclerosis, while platelet-type 12-LOX has been implicated in diabetes, blood coagulation, psoriasis, and cancer. Human reticulocyte 15-lipoxygenase-1 (12/15-LOX, aka 15-LOX-1) is also an attractive therapeutic target, particularly for its role in atherogenesis, diabetes, Alzheimer's, newborn periventricular leukomalacia, breast cancer and stroke (Tong, W. G., et al., Clin. Can. Res. 2002, 8, 3232-3242; Pratico, D.; Zhukareva, V., et al., Am. J. Pathol. 2004, 164, 1655-1662).
The 12/15-LOX has historically been called 15-LOX, 15-LOX-1, or 15-LO-1 in humans, L-12-LOX, leukocyte-type 12-LO, or L-12-LO in mice. The number prefix reflects the preference for 15-HETE in humans, vs. mostly 12-HETE for the mouse 12/15-LOX. The gene in both organisms is termed ALOX15, reflecting the close homology (around 78%) and functional equivalence in both species. Mutation of a key residue in the active site of the rabbit 12/15-LOX which normally generates mostly 15-HETE, switches the product ratio towards the 12 product (Borngraber et al., J. Biol. Chem. 1999, 274, 37345-37350). The variability in the product spectrum thus reflects differences in the active site binding pocket, which is otherwise similar in most lipoxygenases, although the orientation of arachidonic acid presumably varies (Schwarz et al., Biochemistry 1998, 37, 15327-15335).
The difficulty in developing inhibitors that target LOX homologues in both species is that they have different substrate specificities and presumably different inhibitor specificities. Thus, existing inhibitors of 12/15-LOX are typically not very selective with regard to other LOX isoforms, and many additionally are strong antioxidants. While this latter fact may not in itself be damaging, the inherent lack of selectivity suggests a much greater potential for off-target effects.
Introducing humanized versions of the drug target into the animal model is one way to meet the challenge of species-to-species variability, but this approach is in most cases not available. It is therefore of great importance to identify bioactive compounds that target a specific pathway both in rodents, and in humans.